This study evaluated the effect of physical and chemical activation on the diffusion time of different concentrations of hydrogen peroxide (HP) bleaching agents through enamel and dentin. One hundred and twenty bovine cylindrical specimens were divided into six groups (n = 20): 20% HP; 20% HP with light activation; 20% HP with manganese gluconate; 35% HP; 35% HP with light activation; and 35% HP with manganese gluconate. The specimens were fixed over transparent epoxy wells with internal cavities to simulate a pulpal chamber. This chamber was filled with an enzymatic reagent to simulate pulpal fluid. The bleaching gels were applied on enamel surface and the image of the pulpal fluid was captured by a video camera to monitor the time of peroxide penetration in each specimen. ANOVA analysis showed that concentration and type of activation of bleaching gel significantly influenced the diffusion time of HP (P < 0.05). 35% HP showed the lowest diffusion times compared to the groups with 20% HP gel. The light activation of HP decreased significantly the diffusion time compared to chemical activation. The highest diffusion time was obtained with 20% HP chemically activated. The diffusion time of HP was dependent on activation and concentration of HP. The higher concentration of HP diffused through dental tissues more quickly.